The present invention relates to a novel cell adhesion protein, a gene coding for the same, a process for preparing the same and a carrier onto which the same is immobilized.
Forming rosette with sheep erythrocytes has been recognized to be one of specific responses of human T-cells. At present, it is understood that the forming rosette of sheep erythrocytes and human T-cells is a binding response due to high affinity of CD2 antigen receptor on a sheep erythrocyte for CD2 antigen on human T-cells (another name: T11 antigen). Any clear answer has not been obtained for the question why human T-cells form rosette with sheep erythrocytes easily. However, there is a possibility that in the structure of a sheep receptor for CD2 antigen itself there exists a function different from the function of human LFA-3, being a receptor for CD2 antigen in human. At present, it is known that the partly determined N-terminal amino acid sequence of a receptor on a sheep erythrocyte for CD2 antigen (SE0 ID NO:3) has about 50% homology with the amino acid sequence of human LFA-3, a receptor for CD2 antigen (refer to Unexamined Japanese Patent Publication No. 150228/1988). However, it is not known what kind of whole structure the receptor on sheep erythrocyte for CD2 antigen has. As to human LFA-3 molecule, it is known that the molecule is classified as a cell adhesion protein belonging to immunoglobulin superfamily (refer to A. F. Williams and A. N. Barclay, Annu. Rev. Immunol. 6, 381, (1988)) and that the molecule is constructed by, from N-terminus, immunoglobulin-like domain 1 (D 1 region), immunoglobulin-like domain 2 (D2 region), transmembrane region (TM region) and cytoplasm region (C region) (refer to B. P. Wallner et al., J. Exp. Med. 166, 923, (1987)). Further, there is also known human LFA-3 molecule which has D1 region and D2 region and binds to a membrane through glycosyl phosphatidylinositol (refer to B. Seed, Nature 329, 840, (1987)). Therefore, in the present specification, a CD2 antigen receptor having a structure of, from N-terminus, D1 region--D2 region--TM region--C region or a CD2 antigen receptor having D1 region and D2 region and binding to a membrane through glycosyl phosphatidylinositol is referred to as "LFA-3". A sheep receptor for CD2 antigen having unknown structure is kept to be referred to as a receptor for CD2 antigen.
A sheep receptor for CD2 antigen has various uses, such as a use as a reagent for detecting T-cells and a use as a ligand for separating T-cells from a mixture of various kinds of cells, because the receptor has high affinity for CD2 antigen of human T-cells. Further, it is known that CD2 antigen participates in various immune responses as functions of T-cells Therefore, a sheep receptor for CD2 antigen which has affinity for CD2 antigen can be used as an immunoregulative agent and more, as a therapeutic agent which targets a tumor of T-cell family or a leukemia cell by utilizing affinity thereof for T-cells.
A sheep receptor for CD2 antigen can be obtained from sheep erythrocytes. As a process for preparing a sheep receptor for CD2 antigen and a derivative of the receptor, there are known a process comprising solubilizing the receptor from sheep erythrocytes by a surfactant and purifying by an affinity-chromatography using antibodies therefore (refer to Unexamined Japanese Patent Publication No. 150228/1988) and a process comprising solubilizing the receptor from sheep erythrocytes by trypsin (refer to T. Kitao et al., J, Immunol. 117, 310, (1976)). However, a amount of the sheep receptor for CD2 antigen is very small on sheep erythrocytes and it is hard work to prepare a large amount of the sheep receptor for CD2 antigen for the above-mentioned uses.
At present, a protein which naturally exists in very small amount can be prepared inexpensively and in large amount by genetic engineering techniques. For preparing the sheep receptor for CD2 antigen by genetic engineering techniques, a necessary gene coding for the sheep CD2 antigen receptor has to be isolated (cloned) first. However, at present, a whole amino acid sequence of the sheep receptor for CD2 antigen is not known. Further, it is not known at all whether the sheep receptor for CD2 antigen is LFA-3 which is a CD2 antigen receptor having the structure of, from N-terminus, D1 region--D2 region--TM region--C region or a CD2antigen receptor having D1 region and D2 region and binding to a membrane through glycosyl phosphatidylinositol, or a receptor of other structures. Therefore, it has been impossible to detect a gene or mRNA coding for sheep LFA-3, to clone the sheep LFA-3 gene and to prepare sheep LFA-3 by genetic engineering techniques.
It can be considered that some processes can be used for cloning of sheep LFA-3 gene. For example, it may be possible, by using of DNA probe (mixed probe) which is deduced from the known N-terminal partial amino acid sequence of a sheep receptor for CD2 antigen (SEQ ID NO: 3) consisting of 29 amino acid residues, to screen a cDNA of sheep LFA-3 from a cDNA library derived from cells in which the sheep LFA-3 gene is expressed. However, the cDNA screened by the mixed DNA probe can not be certified to be a true gene coding for sheep LFA-3 unless the cDNA is sequenced. A mixed DNA probe is not appropriate for detecting for the sheep LFA-3 gene. A DNA probe which is designed and prepared according to the Lathe et al.'s method (refer to Lathe et al., J. Molec. Biol. 183, 1, 1985) is not always useful to clone a gene.
A monoclonal antibody for a sheep receptor for CD2 antigen has been obtained. It may be possible that cloning of the desired gene can be attained by using the antibody labeled by a radioactive substance to screen the gene from a gene expression library. However, there is no report wherein the monoclonal antibody can be really used for cloning.
Therefore, a sure means by which the sheep LFA-3 gene can be cloned has not been accomplished at present. A DNA probe which is useful for sure cloning of the sheep LFA-3 gene is a DNA probe which has the sequence of LFA-3 gene as it is. Such a DNA probe can selectively hybridize with the sheep LFA-3 gene or mRNA, therefore it is very useful to detect the LFA-3 gene or mRNA.
On the other hand, until now, there have not been known the existence of a LFA-3 like protein deficient in D2 region and a LFA-3 like protein deficient in TM region in sheep and also in humans. Such proteins have been first found in the present invention.
If these proteins have high affinity for CD2 antigen of human T-cells, it is considered that they are useful as a detecting reagent for human T-cells, as a ligand for separating T-cells of humans and the other animals, as an immunoregulative agent or as a therapeutic agent which targets tumors of T-cell family. For these uses, it is necessary to make a mass production of the protein possible. Although a process by genetic engineering techniques is appropriate for a mass production of such a protein, a gene coding for the protein has to be cloned and has to be analyzed in order to perform the process. Further, it is also necessary to search a protein which is more suitable for a process by genetic engineering techniques and to clone a gene coding for such a protein and to analyze the structure of the protein.
An object of the present invention is to provide a protein, which is appropriate for a process by genetic engineering techniques, having high affinity for CD2 antigen on human T-cells.
Another object of the present invention is to provide a gene coding for such a protein.
A further object of the present invention is to provide a process for preparing such a protein by genetic engineering techniques.
A still further object of the present invention is to provide a carrier onto which such a protein is immobilized.
These and the other objects of the present invention will become apparent from the description hereinafter.